In the treatment of diabetes mellitus insulin preparations derived from porcine or bovine insulin have generally been used. Bovine, porcine, and human insulins exhibit minor differences with respect to their amino acid sequence, the difference between human and porcine insulin being confined to a single amino acid in that the B30 amino acid of human insulin is threonine, whereas that of porcine insulin is alanine. However, it could be argued that the ideal insulin preparation for human beings would be an insulin having exactly the same chemical structure as that of human insulin.
For the production of natural human insulin the necessary amount of human pancreas glands is not available.
Synthetic human insulin has been prepared on a small scale at great expense, vide Helv. Chim. Acta 57, 2617, and 60, 27.
Semi-synthetic human insulin has been prepared from porcine insulin by what are believed to be a tedious pathway, vide Hoppe-Seyler's Z. Physiol. Chem. 357, 759.
One known semi-synthetic process for preparing human insulin comprises the following three steps: First, porcine insulin is converted into porcine des-(Ala.sup.B30)-insulin by treatment with carboxypeptidase A, vide Hoppe-Seyler's Z. Physiol. Chem. 359, 799. In the second step, porcine des-(Ala.sup.B30)-insulin is subjected to a trypsin-catalyzed coupling with Thr-OBu.sup.t, whereby human insulin Thr.sup.B30 -tert-butyl ester is formed. Finally, said ester is treated with trifluoroacetic acid yielding human insulin, vide Nature 280, 412. The first step, however, results in a partial removal of Asn.sup.A21, yielding des-(Ala.sup.B30, Asn.sup.A21)-insulin. This derivative gives, after the two subsequent reactions, rise to a contamination by des-(Asn.sup.A21)-insulin in the semi-synthetic human insulin product, a contamination which cannot easily be removed with known preparative methods. Des-(Asn.sup.A21)-insulin possesses low biological activity (about 5%), vide Amer. J. Med. 40, 750.
Direct conversion of porcine insulin into human insulin by traspeptidation was suggested in U.S. Pat. No. 3,276,961, but the process suggested employs conditions under which splitting of the Arg.sup.B22 -Gly.sup.B23 takes place, vide J. Biol. Chem. 236, 743.
Related U.S. patent application, Ser. No. 233,051 filed Feb. 10, 1981, now U.S. Pat. No. 4,343,898 relates to a transpeptidation process for preparing semi-synthetic human insulin from porcine insulin via a threonine B30 derivative. High yields are obtained by the process of this related patent, and in all respects, the patent process is believed to be well adapted to conversion of porcine insulin into human insulin.
The present invention relates to an alternative process which might be competitive with the process of related U.S. Pat. No. 4,343,898. Surprisingly, it has been found that the reaction conditions and reactants suited to the transpeptidation of porcine insulin are applicable to amidation of porcine des-(Ala.sup.B30)-insulin into the threonine.sup.B30 esters of human insulin. Specifically, the method herein described results in yields of threonine.sup.B30 esters of human insulin exceeding 90%.
A like amidation process for preparing human insulin from porcine des-(Ala.sup.B30)-insulin has been described in European patent application No. 80,101,966, vide also Nature 280 (1979), 412, and U.S. Pat. Nos. 4,320,196 and 4,320,197. According to the U.S. patents the amidation is preferably performed in a medium containing 0 to 65%, preferably 40 to 60%, of an organic solvent. Furthermore, the preferred reaction temperature is between 20.degree. C. and 40.degree. C., the temperature 37.degree. C. being used in the examples. The yield of coupling was by HPLC (high pressure liquid chromatography) from 50-80%. According to the Nature paper, the yield was 73%.
An amidation process for preparing human insulin from porcine des-(Ala.sup.B30)-insulin has been described also in Proceedings of the 2nd International Insulin Symposium, Aachen, Federal Republic of Germany, 1979. According to said paper, the amidation was performed in a medium containing about 60% of organic solvent and the reaction was performed at 38.degree. C. The yield of coupling was determined by HPLC to be 67%.
An amidation process for preparing human insulin from porcine des-(Ala.sup.B30)-insulin has been described also in Proceedings of the 16th European Peptide Symposium, Helsingor, Denmark, 1980. According to said paper, the process was performed in a medium containing about 60% organic solvent. Probably, the reaction temperature was 37.degree. C. After a reaction time of 30 minutes, the yield was 85%, however, the yield was decreased to 70% after 22 hours.
One of the reasons for the low yields by the amidation processes known heretofore is the loss of insulin due to undesired side reactions, e.g., forming DOI-Thr(R.sup.2)-R.sup.1, wherein DOI represents porcine des-octapeptide-(B23-B30)-insulin; R.sup.1 and R.sup.2 represent whatever carboxy and hydroxyl protecting group is present on the threonine moiety.
The objective of the present invention was to discover process conditions under which the yield of reaction product is extremely high, specifically, is higher than 90%.
Surprisingly, yields exceeding 90% are obtainable by the use of a much lower concentration of water in the reaction mixture than by the known amidation processes.
Preferably, the amidation is carried out at lower temperatures such as at below room temperature, i.e., at 25.degree. C. or less.
The object of this invention is to provide a process for converting human des-B30-insulin into a threonine B30 ester of human insulin in yields exceeding 90%.